The surf clam, Spisula solidissima, is one of the relatively new seafood resources of the Atlantic coast shellfish processing industry. The major industry growth occurred in the mid 1940's and in 1958, when new offshore beds near Long Island were discovered (Zall et al., 1976, Pro. 7th Natl. Sympos. Food Processing Wastes, Environmental Protect. Series EPA-6002-76-304, Dec. 1, 1942). Today annual amounts of surf clam meats from New York approximate 2,000 metric tons of finished product or about 7% of the total landing of this clam in the United States.
Clam processing consists mainly of shucking, washing, mincing and canning or freezing of the meat. An appreciable amount of wastes, including clam wash water, bellies and shells are generated along with the clam meat in the processing line. Growing public environmental concern has stimulated research to improve the handling and disposal of these wastes. Hood, Zall and Conway (1976, supra) developed a process now being used by the seafood industry to convert minced clam wash water into clam juice. Zall and Cho (1977, Trans. A.S.A.E., 20: 160) showed that additional 5% of meat material could be captured from shell waste m=by manually culling visible amounts of meat adhering to the shell.
The clam belly which constitutes from 7 to 25% of the total meat is currently underutilized and causes a disposal problem. With the support from the New York State Sea Grant Program, this study was initiated to upgrade the economic value of clam belly waste. One hypothesis was that the structure and function of enzymes in the clam might have evolved differently from those of microorganisms, plants or animals encountered terrestrially. Some clam enzymes might have special characteristics suitable for industrial use or be an interesting product for scientific applications. For example, Shallenberger and co-workers (1974, Experimentia, 30: 597) found that the greatest activity of carbohydrases in surf clam was 3)-.beta.-D-glucanase (laminarinase, laminaranase) capable of hydrolyzing the marine polysaccharide laminarian to simple sugars such as laminarabiose and glucose.
Proteases are of increasing importance in industrial applications. The possible uses of proteases include leather-bating, laundry cleaning, silk-degumming, chill-proofing of beer, meat tenderizing, cheese-making and the production of pharmaceuticals. Much information is currently available for the proteases found terrestrially in plant, microbes and animals. However, the select types of properties and functions of proteases which may be found in clams are still much unknown. The first goal of this study was to isolate and characterize dominant proteolytic enzymes which might be found in clam viscera, because only after some basic biochemical properties and functions of the enzymes are understood can scientists predict or put into place uses for novel enzymes discovered. The next goal was to selectively seek the application for defined enzymes on the basis of the acquired information. These objectives were realized and fulfilled during the course of this work: Two proteinases were isolated and characterized from clam processing waste. One was a mammalian cathepsin B-like enzyme. Some potential uses for these two enzymes in processing food were demonstrated, such as in making cheddar cheese and tenderizing meat.
Previous studies on the proteolytic enzymes of invertebrates have focused on the enzymes active primarily at neutral or alkaline pH. Bundy and Gustafson (1973, Compar. Biochem. & Physiol., 44B: 241) purified a trypsin-like protease from the starfish. Trypsin and a low molecular weight protease had been isolated from the cardia fluid of crayfish (Zwilling and Neurath, 1981, Methods in Enzymology, 80: 633). Kozlovskaya and Voskovsky (1970, Compar. Biochem. & Physiol., 34: 137) surveyed a variety of marine invertebrates, including 14 bivalves, for alkaline proteases. A range of results from zero to low activity were obtained for the bivalves. Reid and Rauchert (1970, Comp. Biochem. & Physiol., 35: 689) demonstrated that tryptic and cymotryptic endopeptidases, carboxypeptidases A and B, and leucine aminopeptidase were present in the gastric juice and diverticula extract of Chlamys hericius. Similar results were obtained from several bivalve species (Reid and Rauchert, 1972, Comp. Biochem. & Physiol., 41A: 887) and two univalve species (Cockburn and Reid, 1980, Comp. Biochem. & Physiol., 65: 275). Although the latter investigations concentrated on alkaline endopeptidases from bivalves, it was traditionally realized that the greater portion of protein digestion occurred intracellularly at acid pH levels in the digestive diverticula, under the influence of enzymes about whose characteristics very little was known. Reid and Rauchert (1976, Comp. Biochem. & Physiol., 54B: 467) found that the intracellular proteolytic activity of the digestive diverticula in Tresus capax was approximately four times that of the stomach and acid proteinases are the most important enzymes. The acid endopeptidases have similar characteristics to vertebrate cathepsin B and D. Cathepsin B diminishes in winter when there is little food available while cathepsin D persists. This observation suggested that cathepsin B has a digestive role, and cathepsin D probably has a primary role, such as turnover of tissue protein which is non-digestive. Two acid exopeptidases, cathepsin A and C, are also present in the digestive diverticula. Surprisingly, two cooper-activated acid proteinases were isolated and crystallized from their zymogen forms of a sea mussel, Mytilus galloprovincialis (Dumitru et al., 1977, Revue Roumaine de Biochim., 14: 95; Dumitru et al., 1978, Comp. Biochem. & Physiol., 59B: 81; Iordachescu et al., 1978, Comp. Biochem. & Physiol., 61B: 119).
The fact that all the studies on molluscans have been limited to the digestive organs has led Aikawa and Aikawa (1982, Biochem. Systematics & Ecology, 10: 175) to investigate the distribution of the lysosomal acid proteinases in the tissues relating to various other functions distinct from nutrition. In 12 molluscans surveyed they found that cathepsin D-type proteinases with an optimum pH around 3 were predominant in various tissues, such as adductor muscle, foot, gill, mantle and midgut gland.